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目的:建立方便、快速、高效的小鼠原代血管平滑肌细胞(vascular smooth muscle cell,VSMC)的分离培养方法,为相关研究提供快速获取原代VSMC的方法技术。方法:分离小鼠主动脉,用Ⅰ型胶原酶消化血管组织去除内皮细胞等杂细胞;将血管切成1mm2大小组织块种植于六孔细胞培养板孔底,用分离培养液诱导原代VSMC细胞的繁殖生长。光学显微镜观察细胞形态特征;免疫组化方法鉴定VSMC特异性蛋白α-SMA的表达;RT-PCR方法检测VSMC特异性蛋白α-SMA和SM22α的mRNA表达水平。结果:本方法分离原代VSMC,3d后可见长梭状VSMC从组织块边缘长出,7d后可以进行传代。光学显微镜观察显示,分离细胞具有平滑肌细胞(smooth muscle cell,SMC)形态特征;免疫组化分析显示,分离细胞α-SMA蛋白表达阳性;RT-PCR分析显示,分离细胞中α-SMA和SM22α在mRNA水平高表达。结论:本实验成功建立了一种简便、快捷、高效从组织块分离培养VSMC的改良方法,为快速获得原代VSMC提供了一种适用的方法技术。
Abstract：Objective:To establish a convenient,fast and efficient method for the separation of original generation vascular smooth muscle cells(VSMC)from mice,and also to provide a technical method to obtain the original generation VSMC quickly.Methods:Separated the mouse aorta,digest the vessel with collagenase TypeⅠsolution for the removal of endothelial cells and other miscellaneous cells which adhere to both inside and outside of the vessel.Cut the vessel into pieces,approximately(1×1)mm2 in size and plant these pieces into the bottom of 6-well plate,induced the original generation VSMC to reproduction and growth with separation medium.Observed the cell morphological characteristics with optical microscope,identified the expression of VSMC specific proteinα-SMA by immunohistochemical method,and detected the VSMC specific gene expression ofα-SMA and SM22αat mRNA level by RT-PCR.Results:It can be observed that long fusiform VSMC was generated from tissue block edge after 3days and could be passaged after 7days using our method to separate the original generation VSMC.The separated cells have the typical morphological characteristics of smooth muscle cells(SMC)observed by optical microscope.Immunohistochemical analysis showed that the expression ofα-SMA protein in the separated cells is positive.It was proved by RT-PCR analysis that the expression ofα-SMA and SM22αat mRNA level in the separated cells is higher.Conclusions:From what has been discussed above,this experiment successfully has established an improved method which can be used to separate the original generation VSMC from tissue pieces quickly and efficiently,and also provid an applicable technical method to quickly obtain original generation VSMC for scientific research.
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基本信息:
DOI：10.13210/j.cnki.jhmu.20140112.071
中图分类号:R329
引用信息:
[1]张正杨,王含彦,易芳,等.小鼠原代血管平滑肌细胞改良分离方法的建立[J].海南医学院学报,2014,20(03):302-306+312.DOI:10.13210/j.cnki.jhmu.20140112.071.
基金信息:
四川省科技厅应用基础项目(2006J13-031)~~
2014-03-28
2014-03-28